While the proteins induced in mammalian cells grown under heat-shock conditions have been identified, their function in the cell is unknown. Experiments designed to assess the function of the individual stress proteins will be pursued using biochemical, immunological, and microinjection techniques. Specifically, it is planned to complete the purification of the major mammalian stress proteins, notably the 28kd and 110kd species, and to determine various properties of the purified proteins. Using the purified proteins as antigens, polyclonal antibodies will be raised and used to determine the location of 28kd and 110kd in both normal and stressed cells. Immunological methods have established that a portion of the major induced mammalian stress protein, 72kd, is present within the nucleus and nucleoli of heat-shocked cells. Isolation of nuclei and nucleoli from normal and stressed cells by biochemical methods and analysis of the individual fractions by immunoelectron microscopy, using the anti-72kd antibody, will be performed to provide details of the nuclear/nucleolar association of 72kd. While the significance of this nucleolar locale of 72kd is not known, preliminary evidence is suggestive of 72kd being part of a ribonucleoprotein (RNP) complex. Attempts will be made, therefore, to isolate and characterize this putative RNP by cell fractionation and biochemical methods. Finally, studies will be initiated to examine the effects on cells of introducing either the purified stress proteins (specifically the 72kd, 73kd, and 90kd species) or their corresponding antibodies into cells by the method of microinjection, metabolic labeling of the injected cells, and analysis of the labeled proteins by two-dimensional gels. Questions relating to the possible autoregulation and thermotolerance rendered by the levels of the stress proteins in the cell will be addressed.